For observation of microorganisms such as bacteria which are present on a test surface but cannot be visually observed, there has heretofore been applied a culture method wherein a microorganism is allowed to form a colony. According to this method, a solid plate medium using an agar and the like is pressed against the test surface to transfer the microorganisms on the test surface to the agar plate medium; microorganisms are cultured as they are under the optimal environment; and the colonies formed on the agar plate medium are counted visually. Examples of this method include a food stamp method using an agar stamp commercially available from Nissui Pharmaceutical Co., Ltd. and membrane filter method.
According to the membrane filter method which uses a commercially available membrane filter capable of trapping microorganisms, the microorganisms are collected by thoroughly wiping a test surface with saline, phosphate buffer and the like and filtering the pooled aqueous solution through a membrane filter and the like to trap the microorganisms on the membrane filter. The obtained microorganisms are sufficiently brought into contact with a liquid medium to allow formation of colonies on the filter, and the colonies are counted.
In addition, Igaku Kensa (Medical Test), vol. 41, No. 3, p. 352 (1992) discloses, as an example of culture method, a film coat method including preparation of a film coated with a medium, bringing same into contact with a test surface and culturing same to harvest the detection target microorganism.
A method such as food stamp method, nevertheless, is often associated with insufficient collection efficiency of microorganisms, since it has weak adhesive strength available for transferring microorganisms by pressing a medium against a test surface, and is poor in reproducibility because of variable water contents of the agar medium. Being commonly problematic in various culture methods, contamination among microorganisms is inevitable, which in turn results in interaction among microorganisms on the medium and precludes pure culture, whereby ultimate identification analysis cannot be performed. What is more, not every microorganism is capable of culture to form colonies on a medium and the culture method is limited to the detection of viable cells alone, exclusive of microorganisms that cannot grow in an ordinary medium; thus the method is associated with possible omission of detection. A material limitation on the culture method is that culture time of 1-2 days or longer is needed, so that a real-time monitoring of microorganism detection is not possible.
In the membrane filter method, moreover, a liquid test object such as an aqueous solution can pass through a filter as it is, but other non-liquid test objects require accumulation of microorganisms by time-consuming cumbersome steps of sampling with a cotton swab, preparation of solutions for washing, and the like. In addition, this method does not elude the above-mentioned defects of the culture method, since the determination of microorganisms is based on the culture and the number of colonies formed on a filter by culture. The film coat method also suffer from various defects of the culture method.
The technique recently developed to detect ATP (adenosine triphosphate) in the cells of microorganisms is only applicable to the microorganisms dispersed in water, so that the method for collecting the microorganisms has been the difficult part. As such, there has not been available an easy and simple method for detection of microorganisms in the prior art techniques.